Utilizing tissue clearing based 3-D imaging to quantitatively study neural regulation of craniofacial mesenchymal stem cells Recent discoveries of bone regulation by the nervous system has led to the identification of new regulatory axes between the nervous system and bone1-3. Various studies indicate that signal from the central or peripheral nervous system directly or indirectly regulates the bone remodeling process1, 2. One technical challenge on studying the nerve-bone interactions is to visualize nerve fibers on or within bones because of their fine size (~1m diameter) and the distant location of the neuron cell bodies. In addition, 2-D information obtained from sections made it difficult or impossible to quantify spatial interactions between complex craniofacial tissues. Development of 3-D imaging based tissue clearing technique provides solutions to these two issues. Tissue clearing technique is a recent breakthrough in the neuroscience field. By rendering tissue transparent, 3-D images of the whole organ can be achieved directly with a confocal microscope without sectioning4. We recently developed a new tissue clearing technique named PEG Associated Solvent System (PEGASOS). The PEGASOS method efficiently clears both hard and soft tissue to high transparency and protects endogenous fluorescence with no loss. The PEGASOS method was initially developed to study the neuron connectome within the brain (manuscript in revision). Pilot experiments suggested that it can also be applied on the craniofacial hard tissue. Skull bone, teeth and femur harvested from adult mice can be cleared to nearly invisible after treatment without losing GFP fluorescence. We were able to acquire the 3-D images of vascular network within the tooth, suture, dura and calvarial bone. We were also able to acquire the 3-D images of nerves within the femur periosteum, tibia bone marrow, suture and periodontal ligamental space. Quantitative analysis of these 3-D images indicated that vasculatures and nerves within craniofacial hard tissue present highly heterogenous distribution pattern. In contrast to previous study, our preliminary results suggested that Gli1+ cells within the suture are closely associated with the vasculatures. In the current proposal, we propose to improve the PEGASOS method for craniofacial hard tissue research. With the spatial information provided by PEGASOS based 3-D imaging, we would like to quantitatively test the hypothesis that craniofacial MSCs are associated with the neurovascular bundle (NVB) and nerves are essential for stem cell migration towards the injury site